Objective

Aggregates of proteins such as amyloid-beta and alpha-synuclein circulate the extracellular space of the brain (ECS) and are thought to be key players in the development of neurodegenerative diseases. The clearance of these aggregates (among other toxic metabolites) is a fundamental physiological feature of the brain which is poorly understood due to the lack of techniques to study the nanoscale organisation of the ECS. Exciting advances in this field have recently shown that clearance is enhanced during sleep due to a major volume change in the ECS, facilitating the flow of the interstitial fluid. However, this process has only been characterised at a low spatial resolution while the physiological changes occur at the nanoscale. The recently proposed “glymphatic” pathway still remains controversial, as there are no techniques capable of distinguishing between diffusion and bulk flow in the ECS of living animals. Understanding these processes at a higher spatial resolution requires the development of single-molecule imaging techniques that can study the brain in living animals. Taking advantage of the strategies I have recently developed to target single-molecules in the brain in vivo with nanoparticles, we will do “nanoscopy” in living animals. Our proposal will test the glymphatic pathway at the spatial scale in which events happen, and explore how sleep and wake cycles alter the ECS and the diffusion of receptors in neuronal plasma membrane. Overall, BrainNanoFlow aims to understand how nanoscale changes in the ECS facilitate clearance of protein aggregates. We will also provide new insights to the pathological consequences of impaired clearance, focusing on the interactions between these aggregates and their putative receptors. Being able to perform single-molecule studies in vivo in the brain will be a major breakthrough in neurobiology, making possible the study of physiological and pathological processes that cannot be studied in simpler brain preparations.